Binding assays using optical resonance of colloidal particies

ABSTRACT

A device and a method enable the rapid, quantitative evaluation of a large collection of ligands for binding affinity with a certain immobilized receptor, the improvements being that binding pan be detected without the need for a label and that binding is carried out in solution phase at a high rate. The instrument has at least two embodiments, one is based on a sensitive absorption photometer and the other on a sensitive light scatter photometer operating at a specific resonance wavelength, λ R , of small, metallic, colloidal particles. The resonance is present in small particles having a complex refractive index with real part n(λ) approaching 0 and imaginary part k(λ) approaching {square root}2 simultaneously at a specific wavelength λ R . The particles are substantially spherical and substantially smaller than λ R . The receptor is immobilized on a suspension of such particles and ligand binding is detected by a change in optical absorption or light scatter at the resonance wavelength.

BACKGROUND OF THE INVENTION

[0001] The present application is based on Provisional Application No.60/096,159 entitled “Binding Assays By Means Of Optical Resonance OfColloidal Particles” filed on Aug. 11, 1998 and claims priority fromthat application; the present application is also a continuation in partof copending application Ser. No. 08/789,211 entitled “HomogeneousBinding Assay” filed on Jan. 23, 1996 and incorporated herein byreference.

[0002] 1. Field of the Invention

[0003] The present application concerns the field of detection of ligandbinding and more specifically pertains to high-speed instruments forautomatically screening large numbers of potentially therapeuticcompounds by measuring their relative binding affinity for variousbiological receptors.

[0004] 2. Description of Related Art

[0005] The methods of genetic engineering have been successful inidentifying the genetic sequence of disease-related cell surfacereceptors and artificially expressing these receptors in substantialquantity and in purified form. Such receptors can also be isolated fromcell preparations. Combinatorial chemistry techniques provide rapidsynthesis of low molecular weight compounds that are potential bindingpartners for isolated or identified receptors. Once created, these vastlibraries of compounds must be screened for relative binding affinity tothe target receptor. High speed machinery has become essential for typeof testing or screening to be accomplished in a timely fashion.

[0006] The traditional approach to screening has been to label eachcompound, for example, with a radioactive moiety, a fluorescent tag, ora luminescent tag and to then determine binding of the labeled compoundto the receptor of interest. For this purpose receptor is generallyimmobilized and the labeled compound is allowed to contact the receptor.After a period of time, unbound compound is washed away and theimmobilized receptors are analyzed for the presence of the bound label.

[0007] There are at least two problems in this approach. First, washingtends to disrupt weak binding that may be important for detectingcompounds with some but not ideal activity. Second, the task of labelingan entire library of perhaps hundreds of thousands of compounds presentsa practical limit to the number of compounds that can be screened. Eachcompound presents a unique problem to be solved for labeling, and notall can be labeled by the same tag. Finally, the presence of the labelcan modify the affinity between the compound and the receptor.

[0008] Therefore, it would be highly desirable to have an instrument anda method that eliminates labeling and yet allows the quantitativeanalysis of the binding affinity between a compound and a receptor. Thiswould remove a fundamental limit to high speed screening of very largelibraries of compounds.

[0009] The present invention achieves this goal by providing aninstrument and a method that detects a change in the opticalcharacteristics of the solid support to which the receptor has beenbound when the receptor is occupied by an unlabeled binding compound.The solid support is a specific type of colloidal particle of less than100 nm in diameter. This small size means that the binding kinetics forthe reaction are similar to solution phase kinetics. The instrumentautomatically mixes the compound and the colloidal suspension ofreceptors, incubates the mixture, then reads the result at the rate ofthousands of tests per hour.

[0010] In the copending application referenced above the presentinventors discovered that optical resonance could be used to detect thecrosslinking of particles. The current invention is not dependent oncrosslinking of particles. As developed below, the present inventiondirectly detects the binding of an unlabeled ligand to a particle.

SUMMARY OF THE INVENTION

[0011] The invention utilizes a specialized type of optically sensitive,sub-micron particle, the surface of which is pre-coated with a monolayerof a specific molecular receptor that can bind solution phase ligands(In this invention, “receptor” refers to a molecule immobilized on theparticle that is capable of binding other molecules that are in freesolution. Furthermore, in this invention, the molecules in free solutionare termed “ligands”). The particle itself is a transducer that directlysenses the binding of ligands to the receptor and creates an opticalsignal indicative of this binding. The particle itself is the bindingsensor, thereby obviating the need to label the ligand.

[0012] The signal transduction properties of the particles used in theinvention depend upon there being an optical light scatter andabsorption resonance at a specific resonance wavelength λ_(R). Such aresonance is present in small particles having a complex refractiveindex wherein a real part n(λ) of the index approaches 0 while animaginary part k(λ) approaches {square root}2 simultaneously at thespecific wavelength λ_(R). (In this invention a small particle is onethat is less than approximately one tenth the wavelength of the incidentlight.) It is known from detailed theories of light scatter, that, whenthe above resonance condition is met, both light scatter and absorptionare substantially greater than predicted by the Rayleigh theory ofsimple light scatter where it is assumed that n and k are constant andnot functions of λ (Bohrens and Huffman). The present inventiondemonstrates the additional surprising result that the intensity ofsmall particle light scatter and absorption at the resonance wavelengthchanges when ligands bind to receptors immobilized on such resonantparticles. The closer the two conditions of n and k are met, thestronger is the resonance, and the more sensitive the receptor-coatedparticles are to ligand binding.

[0013] Normally, expressions for light scatter and absorption from smallparticles with constant refractive indices are simple functions ofwavelength. Generally speaking, both light scatter and absorption arehighest for short wavelengths (e.g. for ultra-violet light) and lowestfor long wavelengths (e.g. red or infrared light). This wavelengthdependence is monotonic, meaning that there is a continuous progressionfrom intense absorption and scatter at short wavelengths to less intenseabsorption and scatter at long wavelengths. Exceptions to this behavioroccur for particles with a complex refractive index that has strongwavelength dependence.

[0014] If the refractive index of the particle varies with wavelength insuch a way that the two conditions; 1). n(λ) approaches 0 and 2). k(λ)approaches {square root}2 are simultaneously met at a specificwavelength λ_(R), then light scatter and absorption increasedramatically in a narrow wavelength band around λ_(R). This departure istermed a resonance. The resonance is delicate, and can be perturbed bythe deposition of chemical agents on the surface of the particle. When alayer of receptors is coated onto the particle, the resonance isaltered; but it is a surprising and important aspect of the presentinvention that when ligands then bind to the receptor layer, there is afurther perturbation of the resonance that can be readily detectedoptically.

[0015] The optical signal pertaining to this invention is detectedeither by absorption or light scatter photometry. The optical resonanceincreases the level of light scatter and absorption by at least a factorof ten above the normal levels of light scatter at the resonancewavelength λ_(R). This enables the optical detection of particles thatare as small as the order of 100 Angstroms (10 nm) at lowconcentrations. Such small particles advantageously diffuse virtually asrapidly as macromolecules in solution. Such rapid particle motioncoupled with an interparticle spacing that is of the order ofmicrometers (μm) means that the ligand receptor surface binding reactionoccurs at nearly maximal rates. Such particle-based reactions should becontrasted to binding reactions that occur on flat surfaces such as whenreceptors are immobilized on the surface of micro-wells. There theligand must diffuse over distances of about a millimeter (1000 μm) ormore to reach the receptor. These latter reactions are slow and impedethe desired high throughput of an automated system for binding reactionscreening.

[0016] The binding reactions and signal transduction in the presentinvention occur in a single, rapid step. This enables binding assays tobe easily automated and carried out at such system throughput rates thatlarge collections of ligands and receptors can be evaluated for bindingaffinity in relatively short periods of time by machines that runcontinuously and benefit from low complexity fluid handling mechanisms.

BRIEF DESCRIPTION OF DRAWINGS

[0017]FIG. 1. represents a schematic of light scatter and lightabsorption detection.

[0018]FIG. 2. shows a schematic diagram of light source stabilizationused in the present invention.

[0019]FIG. 3. illustrates the use of optical imaging to minimizebackground scattered light.

[0020]FIG. 4. is a parametric plot of k versus n for silver showing theeffect of resonance on light extinction.

[0021]FIG. 5. is a plot showing the effect of particle diameter onoptical resonance.

[0022]FIG. 6. is a plot showing the effect on optical resonance ofcoating the resonant particles with protein.

[0023]FIG. 7. is a plot showing the effect on optical resonance of thebinding of unlabelled ligand to resonant particles.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The following description is provided to enable any personskilled in the art to make and use the invention and sets forth the bestmodes contemplated by the inventor of carrying out his invention.Various modifications, however, will remain readily apparent to thoseskilled in the art, since the general principles of the presentinvention have been defined herein specifically to provide a method anddevice for detecting the binding of unlabeled ligand to opticallyresonant colloidal particles.

[0025] Referring to FIG. 1, there is a container 10 for the liquidelements of the reaction mixture includes a window 12 for the admissionof light from a light source 14 and a second set of windows 16, 18through which transmitted and scattered light, respectively, can pass toa detector or set of detectors 22, 24. In one embodiment, the containeris a well in a multi-well titer tray and in an alternative embodimentthe container is a cuvette such as is employed in opticalspectrophotometers. One embodiment of the device uses an integratingsphere 26 to integrate the scattered light which is sample through aport in the sphere 26. Other ports allow illumination of the samplecontainer 10 and measurement of the transmitted light by thetransmission detector 22.

[0026] The measurement of light transmission can be carried out bycomparing a sample signal with a reference signal. The sample signal isobtained by measuring light transmitted through the sample at or nearthe resonance wavelength, λ_(R). The reference signal is derived in oneof two ways. Either it comes from light transmitted through the sampleat wavelengths well outside the resonant band at λ_(R) or from lighttransmitted through another well containing particles at the sameconcentration as the reaction mixture but lacking the ligand (e.g., acontrol mixture). Most commonly some sort of optical chopper or,alternatively, an optical beamsplitter is used to derive the sample andreference beams from a single light source. These approaches areimportant when the reacting mixture contains colored components thatcould make the relatively small resonance signal hard to detect withoutsuch a dual beam approach. As shown in FIG. 2 the light source 14 usedfor these measurements is best stabilized. A particularly useful lightsource is provided by a light emitting diode (LED) 14′ that can bedirectly electrically modulated by a light source power module 42 inresponse to a feedback signal from a detector 44 that monitors light atλ_(R) that has not passed through the reaction well.

[0027]FIG. 3 shows a useful optical arrangement for avoiding unwantedbackground signals. A lens 32 images the liquid interior 52 of thereaction chamber 10 onto the surface of an optical detector 22. In thisway the walls 54 of the container 10, which tend to scatter fight andproduce an unwanted background, are defocused and the background lightfrom the walls can be reduced with field stops and/or an aperture 34.

[0028] In this example resonant particles were prepared from silvercolloids, such as can be purchased from British Biocell, International,Cardiff, U.K. Small, silver particles exhibit an optical resonance inthe violet region of the spectrum. FIG. 4A shows a plot of n(λ) and k(λ)that illustrates that n(λ) approaches 0 and k(λ) approaches {squareroot}2 at λ_(R)=390 nm ( FIG. 4B) when the particles are in vacuum. Whenthe particles are immersed in water, the resonance is perturbed andshifted to a longer wavelength near 400 nm. Such resonant behavior isnot seen in macroscopic silver material, it is only seen with smallparticles of silver where the particle boundaries are separated bydistances that are small compared to the wavelength of the incidentlight.

[0029] The resonance wavelength is also a function of particle diameter.FIG. 5 shows a series of light transmission spectra of suspensions ofsilver particles of various diameters taken near the resonancewavelength. It can be seen that, for 20.1 nm diameter particles, theabsorption of the sample increases dramatically over a narrow band near400 nm. The resonance is shifted toward longer wavelengths as theparticle diameter increases, and the resonance becomes less sharplypeaked and more dispersed over a wider wavelength band. For example,with 76.8 nm diameter particles, the resonant wavelength has moved outto about 460 nm and the resonance is about one third as strong as for20.1 nm particles.

[0030] The silver particles were then coated with the binding proteinstreptavidin (MW 65,000). Coating was performed by passive adsorption atroom temperature. For the purpose of protein coating an aqueous solutionof colloidal silver particles was first diluted with Tris/HCl buffer, pH9.0 to an optical absorbance at the resonant wavelength peak of 1.1absorbance units, thereby achieving a final buffer concentration of 200mM Tris/HCl. Streptavidin was added to a concentration of 0.1 μg/ml andincubated for 20 min at room temperature. Spectrophotometricmeasurements were taken before the addition of protein and 20 min afterthe addition of protein. For purposes of absorption reading both sampleswere diluted 1:10 with 200 mM Tris/HCl buffer, pH 9.0. FIG. 6 shows theparticle absorption resonance 62 before and after 64 protein coating.The Streptavidin perturbs the resonance by shifting the absorption peakvery slightly to a longer wavelength and lowers the absorption maximum.

[0031] When Streptavidin-coated particles were later contacted with asolution of biotin, the resonance peak was observed to decrease overtime. FIG. 7 is an absorption spectrum 68 taken five minutes after theStreptavidin coated particles were contacted with an aliquot of asolution containing 10 ng/ml of biotin (MW 100). The binding reactionwas essentially complete within five minutes. The time course of biotinbinding can be measured directly in the reaction mixture. This iscompared with an absorption spectrum 66 of the particles prior to theaddition of biotin (this sample was pre-diluted with an aliquot ofbuffer equal to the later biotin addition to correct for dilutioneffects.

[0032] The decrease in the resonant absorption peak after five minutesof binding is a function of the biotin concentration as shown in thetable below. Biotin Concentration Absorbance Decrease  1 nanograms/ml0.002 absorbance units decrease  3 nanograms/ml 0.010 absorbance unitsdecrease 10 nanograms/ml 0.015 absorbance units decrease

[0033] These Streptavidin-coated particles were tested for specificityby contacting them with other small molecules that do not bind toStreptavidin. In all cases, the absorbance change was less than 0.001absorbance unit up to concentrations of 10 micrograms/ml.

[0034] In a second test, the solution phase hapten rhodamine was usedwith a murine anti-rhodamine antibody immobilized on the silverparticles. The observations of a similar decrease in optical absorbanceover a similar time were repeated, indicating that the molecular weightand size differences between Streptavidin and mouse immunoglobulin donot significantly affect the outcome of the experiment.

[0035] Other methods of immobilizing biomolecules on the surface of thesilver or other resonant colloidal particles are possible. Large numbersof bifunctional or polyfunctional organic molecules with reactive groupsfor attaching biomolecules to solid surfaces are well-known to those ofskill in the art. These reactive groups include aldehydes, isocyanates,isothiocyanates, and succinimidyl esters among others. Besides simpleabsorption self-assembling monolayers can also be used to providefunctional groups at a metal surface that are useful in immobilizingreceptors. Receptors that are isolated from cells can also beimmobilized together with fragments of cell membrane in which thereceptors are embedded.

[0036] An actual instrument based on the principles of the presentinvention would include some type of sample handling fluidics and dataprocessing system (computer) as well as the spectrophotometers describedabove. For example, in a screening of a chemical library looking for anew therapeutic drug an aliquot of each compound to be screened could beplaced in one well of a multi-well titer plate. An automatic indexingsystem would move the plate to advance each well into the analyzing beamof a spectrophotometer as defined above. As the well advances into theanalysis position an automatic pipetting system dispenses an aliquot ofcolloidal particles into the well. The particles have on their surfacesthe receptor with which the candidate drug is to interact. The pipettingsystem or some other automatic device insures that the particles and thetest compound are thoroughly mixed. The spectrophotometer then measuresany changes at the resonant peak as an indication of binding. Preferablythese measurements take place over a few minutes so that a time courseof binding can be determined. The computer receives this data andcalculates the results for each candidate compound. The device thenmoves on to the next well. If binding is measured over a five minuteperiod, 12 sample compounds can be analyzed per hour. If the test periodis shorter, a larger number can be screened. An instrument can bepreloaded with dozens of plates so that analysis can proceed day andnight without human intervention. The instruments are small and fairlyeconomical so that a plurality of instruments can operate in parallel toscreen thousands of candidate compounds in a 24 hour period. Of course,many test variations are possible. By including a known ligand for thereceptor one can screen for competition or inhibition by the testcompound.

[0037] In addition to the equivalents of the claimed elements, obvioussubstitutions now or later known to one with ordinary skill in the artare defined to be within the scope of the defined elements. The claimsare thus to be understood to include what is specifically illustratedand described above, what is conceptually equivalent, what can beobviously substituted and also what essentially incorporates theessential idea of the invention. Therefore, it is to be understood that,within the scope of the appended claims, the invention may be practicedother than as specifically described herein.

We claim:
 1. A method for determining binding of a ligand comprising thesteps of: providing colloidal particles having a complex index ofrefraction and showing optical resonance at a resonant wavelength, saidparticles coated with a receptor for the ligand, wherein the resonantwavelength is a wavelength at which a real part n(λ) of the index ofrefraction approaches zero while an imaginary part n(λ) of the index ofrefraction simultaneously approaches {square root}2, and whereindiameter of said particles is less than about {fraction (1/10)} of theresonant wavelength; contacting the colloidal particles with a samplesolution; and measuring light scatter and/or light absorption at theresonant wavelength whereby a decrease in light scatter and/or lightabsorption at the resonant wavelength is representative of binding ofthe ligand to the colloidal particles.
 2. A system for determiningbinding of a ligand comprising: a sample container; colloidal particleshaving a complex index of refraction and showing optical resonance at aresonant wavelength, said particles coated with a receptor for theligand, wherein the resonant wavelength is a wavelength at which a realpart n(λ) of the index of refraction approaches zero while an imaginarypart n(λ) of the index of refraction simultaneously approaches {squareroot}2, and wherein diameter of said particles is less than about{fraction (1/10)} of the resonant wavelength; means for contacting thecolloidal particles with a sample liquid to form a mixture thereof;means for placing the mixture into the sample container; and means formeasuring light absorption and/or light scatter of mixture in the samplecontainer at a resonant wavelength, whereby a decrease in light scatterand/or light absorption at the resonant wavelength is representative ofbinding of the ligand to the particles.
 3. A method for determiningbinding of a ligand comprising the steps of: providing colloidal silverparticles coated with a receptor for the ligand, said silver particleshaving a diameter less than about 70 nanometers; contacting thecolloidal particles with a sample solution; and measuring light scatterand/or light absorption at a resonant wavelength between 350 and 500nanometers whereby a decrease in light scatter and/or light absorptionat the resonant wavelength is representative of binding of the ligand tosaid colloidal particles.
 4. A system for determining binding of aligand comprising: a sample container; colloidal silver particles coatedwith a receptor for the ligand and having a diameter of less than about70 nanometers; means for contacting said particles with a sample liquidto form a mixture thereof; means for placing the mixture into the samplecontainer; and means for measuring light absorption and/or light scatterof mixture in the sample container at a resonant wavelength between 350nanometers and 500 nanometers, whereby a decrease in light scatterand/or light absorption at the resonant wavelength is representative ofbinding of the ligand to the colloidal silver particles.